Acute liver failure is a clinical syndrome that results from massive necrosis and apoptosis of liver cells leading to hepatic encephalopathy and severe impairement of hepatic function. It is caused by different kinds of diseases, such as viral hepatitis (A, B, C, . . . ), drugs, intoxication, auto-immune hepatitis, etc. Many studies have shown that TNF plays a central role in liver disease. TNF is produced mainly by activated macrophages but is also produced in smaller amounts by several other cell types. TNF exerts a variety of effects on different cell types and is implicated as an important mediator in various physiological and pathophysiological conditions. In addition, it has become clear that TNF is an important mediator of apoptosis (programmed cell death).
TNF was originally identified by its capacity to induce hemorrhagic necrosis of tumors in mice. Attempts to use TNF for systemic anti-cancer therapy have failed due to the appearance of severe side effects before therapeutic doses could be reached. One of the side effects of TNF treatment was an elevation in serum levels of transaminases and bilirubin levels, indicating a direct cytotoxic effect of TNF on human hepatocytes. Subsequent studies have shown that TNF may be involved in viral hepatitis, alcoholic liver disease, and fulminant hepatic failure (Muto et al., 1988; Bird et al., 1990; Gonzalez-Amaro et al., 1994; Diehl et al., 1994; Larrea et al., 1996). TNF serum levels are clearly elevated in patients with fulminant hepatitis (Muto et al., 1988). In addition, it was found that serum TNF levels were significantly higher in patients who died than in patients who survived (Bird et al., 1990).
A role for TNF in the pathogenesis of chronic hepatitis B and C viral infection has been suggested. Both viruses induce TNF expression in human liver and human hepatoma cell lines (Gonzalez-Amaro et al., 1994). Patients with chronic hepatitis B have elevated plasma TNF levels, and their peripheral blood mononuclear cells show enhanced TNF production in vitro. In addition, in chronic hepatitis B-infected patients undergoing interferon treatment, a massive increase in spontaneous TNF production by blood mononuclear cells was observed at the time of successful antigen seroconversion (Diehl et al., 1994), suggesting that the increased TNF levels may be involved in hepatitis B virus clearance. Furthermore, the serum levels of soluble TNF-R1 and TNF-R2 are significantly elevated in chronic hepatitis B infection. The serum levels of soluble TNF-R2 correlate closely with the extent of inflammation and hepatocyte death in the liver. During interferon therapy, the response and the increase in transaminases are associated with an increase in soluble TNF-R2 serum levels. For hepatitis C patients, interferon treatment clears the virus and reduces TNF levels to normal in responsive patients (Larrea et al., 1996). Interestingly, pretreatment levels of TNF were higher in unresponsive compared with responsive patients (Larrea et al., 1996). Hepatitis C proteins interact with the TNF receptor, although whether this interaction promotes or prevents apoptosis is not clear (Ray et al., 1998). Recently, an interaction between hepatitis C virus NS5A protein and the TNF-receptor-associated proteins TRADD and TRAF2 has been shown (Majumder et al., 2002; Park et al., 2002). Park and coworkers showed that NS5A impairs TNF-mediated hepatic apoptosis by preventing the association between TRADD and FADD. Moreover, both groups also showed that NS5A prevents TRADD and TRAF2-mediated NF-κB activation.
TNF serum levels are increased in patients with alcoholic hepatitis, and the levels correlate inversely with patient survival. TNF concentrations were significantly higher in patients who did not survive an episode of acute alcoholic hepatitis (Bird et al., 1990). Monocytes isolated from patients with alcoholic hepatitis spontaneously produced higher amounts of TNF compared with healthy controls. Monocytes derived from patients with alcoholic hepatitis also produced significantly more TNF in response to LPS than normal monocytes. Several hypotheses have been developed to explain increased TNF levels in patients with chronic ethanol exposure. Chronic ethanol feeding increases the permeability of the gut to bacterial products such as LPS, potentially inducing TNF production in macrophages (McClain, 1991). In addition, studies investigating the promoter polymorphism in patients with alcoholic steatohepatitis indicated that patients with alcoholic steatohepatitis had a mutation in the TNF promoter that increases its activity (Grove et al., 1997). Thus genetic factors may be involved in the increased TNF production in patients with alcoholic hepatitis.
The role of TNF in liver injury has been studied in several animal models. By using neutralizing anti-TNF antibodies or knockout mice for TNF, TNF-R1, or TNF-R2, it has become evident that TNF triggers apoptosis and/or necrosis of hepatocytes in vivo. In different animal models of liver injury, TNF plays a central or an additive role in the pathogenesis of acute liver injury. Here we used the TNF/Galactosamine (GalN) model. In this model, TNF is administered in combination with D-(+)-galactosamine (GalN), a hepatotoxin, that selectively blocks transcription in hepatocytes by depleting uridine nucleotides (Dekker and Keppler, 1974), inducing lethality, activation of caspases and subsequent hepatocyte apoptosis (Leist et al., 1995; Van Molle et al., 1999; Tiegs et al., 1989). TNF-R1 knockout mice are resistant to TNF/GalN treatment, demonstrating the essential role of TNF-R1 in this apoptosis model (Leist et al., 1995). The sensitizing effect of GalN suggests that the transcriptional block induced by GalN directly inhibits synthesis of anti-apoptotic proteins. Recently, the transcription factor NF-κB has been shown to regulate the expression of a number of anti-apoptotic proteins.
NF-κB is an essential transcription factor that is ubiquitously expressed in all cell types and whose activity is modulated by a wide range of inducers, including cytokines and bacterial or viral products. Many of the NF-κB responsive genes play a key role in the regulation of inflammatory and immune responses. Deregulation of NF-κB activity is often observed in several chronic inflammatory diseases such as rheumatoid arthritis, asthma and inflammatory bowel disease, as well as in acute diseases such as septic shock. Furthermore, NF-κB serves to protect against apoptosis and supports cell cycle progression. The first indication that NF-κB activation may modulate hepatocyte responses relevant to liver injury was the finding that knockout mice deficient in the p65/Rel-A subunit of NF-κB were nonviable because of massive hepatocyte apoptosis during embryogenesis (Beg et al., 1995). Recent reports from several laboratories have now demonstrated that NF-κB activation regulates hepatocyte proliferation and apoptosis in vivo and in vitro. In rats subjected to partial hepatectomy, inhibition of NF-κB activation impaired subsequent liver regeneration and triggered hepatocyte apoptosis (Iimuro et al, 1998). These findings suggest a critical role for NF-κB activation in hepatocytes following a mitogenic stimulus, although the mechanism by which inhibition of NF-κB activity blocked proliferation is unclear. Apoptosis may have resulted from a cell cycle block or from sensitization to TNF produced following partial hepatectomy. An essential role for NF-κB activation during hepatocyte proliferation is also supported by the finding that inhibition of NF-κB activity resulted in apoptosis in an exponentially growing murine hepatocyte cell line (Bellas et al., 1997). However, other studies in confluent rat hepatocyte cultures have demonstrated that NF-κB inhibition by itself did not result in cell death (Xu et al., 1998). In these cells, NF-κB inhibition did convert the hepatocellular response to the mitogenic stimulus of TNF from proliferation to one of apoptosis (Xu et al., 1998). The mechanism by which NF-κB inactivation triggered TNF-induced apoptosis in these studies involved activation of the caspase cascade, and cell death could be prevented by caspase inhibition or NO (Xu et al., 1998).
The NF-κB-dependent gene product(s) that protects hepatocytes against TNF-induced injury remains to be identified. Possible candidate genes are iNOS and interleukin-6, since they are regulated by NF-κB and their gene products may have hepatoprotective effects. It also remains to be determined whether NF-κB activation inhibits hepatotoxicity from injurious agents other than TNF. In the hepatoma cell line Hep G2, treatment with a nontoxic concentration of the superoxide generator menadione protected against subsequent toxic doses of menadione or H2O2 by an NF-κB-dependent mechanism (Chen and Cederbaum, 1997). However, studies in a rat hepatocyte cell line demonstrated that, although H2O2 and copper induced NF-κB activation and caused apoptosis at toxic concentrations, inhibition of NF-κB activity did not sensitize the cells to death from H2O2 or copper (Xu et al., 1998). NF-κB activation may therefore stimulate a defense mechanism specific for the TNF death pathway.
The possibility that NF-κB activation in hepatocytes is protective following liver injury points to the complexity of events following global activation of NF-κB in all cell types in the liver. After a toxic stimulus, it is known that activation of NF-κB in hepatic macrophages results in the production of injurious products such as cytokines and reactive oxygen intermediates. Inhibition of hepatic NF-κB activation was therefore viewed as a potential therapy for liver injury. It now appears that NF-κB signalling represents a problematic therapeutic target, since blanket inhibition of hepatic NF-κB activation may lead to both beneficial and detrimental effects.
Recently, considerable progress has been made in understanding the details of signalling pathways that regulate and mediate NF-κB activation in response to TNF and IL-1. These cytokines act by binding to specific cell surface receptors, which in turn initiate the recruitment of a number of specific adaptor proteins, and the activation of a kinase complex that phosphorylates the NF-κB inhibitor IκB. The latter retains NF-κB in the cytoplasm in an inactive dimeric form. Once phosphorylated, IκB is marked for ubiquitination and subsequent degradation by the proteasome, allowing the nuclear translocation of NF-κB. Whereas members of the IκB family have been well studied as direct inhibitors of NF-κB, a number of other proteins have been reported to negatively regulate NF-κB-dependent gene expression. We and others have previously shown that the zinc finger protein A20 is a potent inhibitor of NF-κB activation in response to TNF, IL-1, LPS and CD-40 (reviewed in Beyaert et al., 2000). In addition, A20 also exerts an anti-apoptotic function in a number of cell lines. A20 is only expressed upon NF-κB activation, and is involved in the negative feedback regulation of NF-κB activation. A20-deficient mice were recently shown to be defective in the termination of NF-κB activation, leading to strong inflammatory responses and cachexia (Lee et al., 2000). The underlying mechanisms responsible for the inhibition of NF-κB-dependent gene expression by A20 is still unclear. A20 interacts with the IκB kinase complex, as well as with TRAF2 and TRAF6, which are part of the IκB kinase activation cascade initiated by TNF and IL-1/LPS, respectively. In addition, three novel A20-binding proteins (ABIN, ABIN-2 and ABIN-3) were recently isolated. Upon overexpression in cell lines, these proteins were shown to inhibit NF-κB-dependent gene expression in response to TNF or IL-1 (Beyaert et al., 2000; Heyninck et al., 1999; Van Huffel et al., 2001, Van Huffel et al., unpublished; AJ320534).